REVERSIBLE IMMORTALIZATION OF MAMMALIAN-CELLS MEDIATED BY RETROVIRAL TRANSFER AND SITE-SPECIFIC RECOMBINATION

A procedure of reversible immortalization of primary cells was devised by retrovirus-mediated transfer of an oncogene that could be subseque ntly excised by site-specific recombination. This study focused on the early stages of immortalization: global induction of proliferation an d life span extension of cell populations. Comparative analysis of Cre /LoxP and FLP/FRT recombination in this system indicated that only Cre /LoxP operates efficiently in primary cells. Pure populations of cells in which the oncogene is permanently excised were obtained, following differential selection of the cells, Cells reverted to their preimmor talized state, as indicated by changes in growth characteristics and p 53 levels, and their fate conformed to the telomere hypothesis of repl icative cell senescence. By permitting temporary and controlled expans ion of primary cell populations without retaining the transferred onco gene, this strategy may facilitate gene therapy manipulations of cells unresponsive to exogenous growth factors and make practical gene targ eting by homologous recombination in somatic cells, The combination of retroviral transfer and site-specific recombination should also exten d gene expression studies to situations previously inaccessible to exp erimentation.